Leukotriene D4 increases nasal blood flow in humans

The effect of leukotriene D4 on the nasal mucosal blood flow in humans was measured using a laser doppler flow meter. The leukotriene solution was applied topically to the nasal mucosa in 9 healthy subjects, and changes in blood flow were measured non-traumatically by the laser doppler instrument. The response showed a consistent increase in blood flow. A dose-response was found in the range of 0.063 - 4.0 nanomole. These results confirm an earlier study on the human skin, implicating leukotriene D4 as an important vasodilator in humans. No increase in nasal secretion was noted by the subjects tested.     

Leukotriene D4 reduces coronary blood flow in the anesthetized dog

We studied the effects of intracoronary administration of leukotriene (LT)D₄ on coronary blood flow and myocardial function in chloralose anesthetized dogs. For comparison, the effects of injections of U-46619 were examined in the same dogs. Both LTD₄ and U-46619 decreased coronary blood flow, left ventricular dP/dt and cardiac output. LTD₄ was ten times more potent than U-46619 in decreasing coronary blood flow. The effects of neither drug were different after indomethacin administration.     

The effect of leucotriene C4 and D4 on cutaneous blood flow in humans

Using a laser-Doppler-flowmeter the microvascular response to LTC₄ and LTD₄ was measured. Intradermal injection of 1 Ug LTC₄ and LTD₄ caused an increase in the microvascular cutaneous bloodflow. The increase in flow was equal to that caused by histamine in equimolar amounts. Blocking the triple-response did not change the response. The values measured after injection of histamine and leucotrienes were about 10–15 times the values found in undisturbed skin and represents probably a maximally dilated vascular bed. Injection of the leucotrienes caused a slight sensation of pain.     

Leukotriene D4-induced hypotension is reversed by thyrotropin-releasing hormone

Injection of leukotriene D₄ (LTD₄, 20 μg/kg, i.a.) to conscious spontaneous hypertensive (SHR) rats produces a short-lasting pressor and tachycardic response followed by prolonged hypotension and bradycardia. Plasma norepinephrine and epinephrine were elevated at the peak pressor/tachycardic phase as well as at the hypotensive phase. Injection of thyrotropin-releasing hormone (TRH, 2 or 5 mg/kg) at the peak of the LTD₄-induced hypotension resulted in prompt reversal of the hypotension and bradycardia in a dose-related manner. Naloxone (5 mg/kg) had no effect on blood pressure and heart rate LTD₄- treated SHR rats. Pretreatment with TRH (5 mg/kg) did not prevent the depressor effect of LTD₄, but attenuated the bradycardic effect of this leukotriene. In addition, TRH had no effect on LTD₄-induced hypotension in the pithed SHR rat. These results suggest that TRH might exert beneficial effects in hypotensive states mediated by leukotrienes or other mediators of anaphylactic reactions.     

Leukotrienes C4 and D4 psoriatic skin lesions

Chemoattractant arachidonate lipoxygenase products have been recovered from the skin lesions of psoriasis, and may play a role in eliciting the intra-epidermal neutrophil infiltrate that characterises this disease. In view of evidence for lipoxygenase activity in psoriasis, the characteristic vasolidation in psoriatic lesions, and the vasodilator properties of leukotriene (LT) C₄ and D₄ in human skin, the presence of these LTs in psoriatic lesions has been investigated. Skin chamber fluid from abraded psoriatic lesions contained significantly greater amounts of immunoreactive material than that from clinically normal skin, as determined by a double antibody radioimmunoassay (RIA) that uses antiserum cross-reacting with both LTC₄ and LTD₄. Purification of lesional chamber fluid and scale extracts by high performance liquid chromatography (HPLC) and RIA of fractions showed immunoreactivity which co-eluted with standard LTC₄ and LTD₄. These findings suggest that LTC₄ and LTD₄ may play a role in mediating the vasodilation and increased blood flow that characterise psoriatic skin lesions.     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B₄, C₄ and D₄ (total dosage of 4 μg/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymp fistulas. In anesthetized as well as unanesthetized sheep, LTB₄ caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow x lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (P ) also increased. In the acutely-prepared sheep, the LTB₄-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C₄ increased P to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Q̇_lym) and lmph-to-plasma protein concentration (L/P) ration in either group. LTD₄ increased P and Q̇_lymp in both acute and awake sheep; Q̇_lym increased without a significant change in the L/P ratio. The LTD₄-induced rise in P occurred in association with an increase in plasma thromboxane B₂ (Txb₂) cocentration. The relativity small increase in Q̇_lym with LTD₄ suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB₄ induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC₄ and LTD₄ cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greather pre-capillary constriction with LTC₄ because Q̇_lym did not change and greater post-capillary constriction with LTD₄ because Q̇ increased with the same rise in P .     

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice

The clearance of human fibrinogen fragments D₁, D₂, D₃ and fibrin fragment D₁ dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D₁ and its dimer were essentially identical. Fragments D₂ and D₃ cleared at a progressively slower rate. Competition studies were performed between ¹²⁵I-labeled fragment D₁ and large molar excesses of unlabelled human fragments D₁, D₂, D₃, D₁ dimer, fragment E, fibrinogen, macroalbumin, mannan and asialooroscomucoid. Of these ligands only the fragment D variants competed for the clearance of ¹²⁵I-labeled fragment D₁. Cross-competition was observed when ¹²⁵I-labeled fragment D₁ dimer was cleared in the presence of large molar excesses of fragment D₁. Autopsies demonstrated that injected fragments D₁, D₂, D₃ and D₁ dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D₁. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cell, respectively). It is concluded that fragments D₁, D₂, D₃ and D₁ dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D₁, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.     

Use of vitamin D4 analogs to investigate differences in hepatic and target cell metabolism of vitamins D2 and D3

In this study, we used molecules with either of the structural differences in the side chains of vitamin D₂ and vitamin D₃ to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems—HepG2 and HPK1A-ras—to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1α-hydroxyvitamin D₂ (1α-OH-D₂) but not for 1α-OH-D₃ is also observed in both 1α-OH-D₄ and Δ²²-1α-OH-D₃ metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22–23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1α,25-(OH)₂D₄, was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D₂ compounds without evidence of side chain cleavage observed for vitamin D₃ derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D₂ and D₃ compounds. Novel vitamin D₄ compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D₄ analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.     

Quantitation of luekotrienes C4, D4 amd E4 released by pathophysiological stimuli

In order to examine the modulation of leukotriene (LT) release, the PAF-acether-mediated stimulation of these compounds in rat lung was studied. Release of LTC₄, LTD₄ and LTE₄ in both perfused and chopped lung preparations was measured using HPLC and radioimmunoassay. Pre-incubation or pre-infusion of the tissue with indomethacin and PGE₂ was conducted to investigate the effect of cyclooxygenase inhibitors and products on the lipoxygenase pathway. In addition, the effects of LT levels of pre-incubation with vasoactive intenstinal polypeptide (VIP) in chopped lung were observed.In perfused rat lung, indomethacin reduced the levels of LTC₄ relative to LTD₄ as measured in the first 2 min after stimulation of the lung by PAF-acether. Chopped lung preparations, incubated for 15 min. exhibited higher levels of LTC₄ and LTD₄ in indomethacin-treated samples, this increases being effectively reversed by PGE₂.In the VIP pre-incubation experiments clear inhibition of peptido -leukotriene synthesis was observed, with no LTC₄ and only low levels of LTD₄ and LTE₄ observed in VIP-incubated samples. In preliminary experiments using rabbit C5a des arg and PAF-acether on rabbit lung parenchyma strips to stimulaet LT release, disodium cromoglycate pre-incubation was observed to inhibit this release.Inhibition of the 5-lipoxygenase pathway of PGE₂ is supported by these experiments. VIP appears to act as an inhibitor of LTC₄ and LTD₄ biosynthesis or release in this model. Too little is known that peptidergic actions to postulate a mechanism by which a neuroendocrine peptide exerts control of release of arachidonate metabolites; however, VIP is associated with muscarinic stimulation (1) and has been found in mast cells (2).     

Autoradiographic localization of leukotriene C4 and D4 binding sites in guinea-pig lung

Binding of [3H] leukotriene C4 and D4 to guinea-pig lung sections was charaterised and binding sites were localized by autoradiography. Both leukotrienes bound to guinea-pig lung sections and membranes with high affinity and with similar charateristics to binding in a membrane preparation. Autoradiography revealed that the distribution of LTC4 and D4 binding sites was markedly different. Smooth muscle and epithelium of central and peripheral airways were densely labelled with [3H]LTC4; vascular smooth muscle and alveolar walls were also labelled. With [3H]LTD4, however, there was no detectable labelling of airways or vessels but subtantial labelling of alveolar walls. This lends futher support that LTC4 and LTD4 binding sites differ and may not be identical with functional receptors.     

Cardiovascular effects of prostaglandin D3 and D2 in anesthetized dogs

Prostaglandin (PG) D₃ has been identified as an inhibitor of human platelet aggregation, but little is known of the hemodynamic activity of this material. In morphine pretreated, chloralose-urethan anesthetized dogs, bolus intravenous injections (1, 3.2 and 10 μg/kg) of PGD₃ and also PGD₂ were associated with marked, dose-related increases in pulmonary arterial pressure. Cardiac index and rate increased, while peripheral vascular resistance decreased in response to injections of PGD₃. A biphasic (depressor followed by a pressor phase) effect on systemic arterial pressure was observed after PGD₂, while PGD₃ was associated with dose-related depressor responses. Graded intravenous infusions (0.25, 0.50 and 1.0 μg/kg/min) of PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses. Quantitatively, PGD₃ infusions were associated with greater decreases in peripheral vascular resistance and greater increases in cardiac output, heart rate, and peak left ventricular dp/dt than were infusions of PGD₂. In contrast, PGD₃ was less potent than PGD₂ as a pulmonary pressor material. Systemic arterial pressure responses to infusions of the prostaglandins were variable. In these experiments, PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses characterized by peripheral vasodilatation.     

Leukotriene B4 potentiates airway muscle responsiveness and

We studied the effects of leukotriene B₄ (LTB₄) on guinea pig airway muscle responsiveness and . Responsiveness in vivo was assessed by measuring specific airway resistance (SRaw) upon intravenous acetylcholine infusion in 5 unanesthetized, spontaneously breathing guinea pigs. We found that aerosolized LTB₄, in a concentration that itself had no effect on baseline SRaw, caused a substantial increase in bronchial reactivity to i.v. ACh within 8 min of its administration. Responsiveness was assessed by measuring isometric contraction of the guinea pig trachealis upon stimulation by either chemical or electrical field stimuli. These studies showed that a concentration of LTB₄ that itself did not cause contraction, potentiated airway muscle contraction to ACh and KCl, but not to norepinephrine. This effect of LTB₄ was substantially reduced by nifedinpine. Our data suggests that amounts of LTB₄ that are themselves non-contractile or in , may directly potentiate the responsiveness of airway smooth muscle to other bronchoconstrictors.     

Detection of leukotriene C4 and D4 in the exudate of rat carrageenin-induced pleurisy

Rat carrageenin-induced pleurisy was used as an acute exudative inflammatory model. The crude ethanol extract of the pleural fluid at 5 hr after carrageenin injection caused the very slow contraction of guinea-pig ileum, which was antagonized by FPL 55712 (1 μg/ml). The ethanol extract was cleaned by LH-20 and was rendered for separation of LTC₄ and LTD₄ by reversed-phase high-performance liquid chromatography (HPLC). Two peaks which showed the same retention time on HPLC as those of LTC₄ and LTD₄ had the contractile activity of guinea-pig ileum and the ratios of the contractile activity to the height on HPLC agreed with those of synthetic LTC₄ and LTD₄. Two peaks of Δ⁶-trans-LTB₄, 5S,12R-(E,E,E,Z)-diHETE and 5S, 12S-(E,E,E,Z)-diHETE, were detected, but the appreciable amount of LTB₄ was smaller than that of each Δ⁶-trans-LTB₄ in the pleural fluid at 5 hr.     

Vasoconstrictor effects of leukotrienes C4 and D4 in the feline mesenteric vascular bed

The effects of leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD₄) in the feline mesenteric vascular bed were investigated under conditions of controlled blood flow so that changes in perfusion pressure directly reflect changes in vascular resistance. Intra-arterial injections of LTC₄ and LTD₄ (0.3–3.0 μg) increased perfusion pressure in a dose-related fashion. Vasoconstrictor responses to LTC₄ and LTD₄ were similar to norepinephrine (NE) whereas mesenteric vasoconstrictor response to the thromboxane analog, U46619, was markedly greater than were responses to LTC₄ and LTD₄. Meclofenamate in a dose that greatly attenuated the systemic depressor response to arachidonic acid was without effect on vasoconstrictor responses to LTC₄ and LTD₄, NE and U46619 in the mesenteric vascular bed. The present data show that LTC₄ and LTD₄ possess significant vasoconstrictor activity in the feline mesenteric vascular bed. In addition, the present data suggest that products of the cyclooxygenase pathway do not mediate vasoconstrictor responses to LTC₄ and LTD₄ in the intestinal circulation of the cat.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4

The pharmacology of leukotrienes (LT) C₄ and D₄ in isolated airway smooth muscle was investigated. In rat trachea, neither LTC₄ or D₄ elicited a response. In contrast, LTC₄ was a potent contractile agonist in guinea-pig trachea, bronchus and parenchymal lung strip. Similar effects were obtained with LTD₄ in trachea and parenchyma. In trachea and bronchus, the concentration-response curve to LTC₄ was biphasic: indomethacin converted the biphasic response curve to a simple sigmoidal shape and enhanced the maximum contractile response. The SRS-A antagonist FPL 55712 antagonized the effect of LTD₄ in both trachea and parenchyma. As regards LTC₄-induced contraction of trachea and bronchus, FPL 55712, depending on concentration, either antagonized, or antagonized and enhanced the maximum contractile response. The enhancement of the maximum contractile response by FPL 55712 was not apparent when indomethacin was present. FPL 55712 failed to antagonize the effect of LTC₄ in parenchyma.     

Secretogogue responses of leukotriene C4, D4: Comparison of potency in canine trachea

By the use of close arterial injection of leukotrienes into the circulation supplying the upper cervical canine trachea, it has been possible to assess the secretogogue effects of leukotriene C₄, and D₄ on mucus secretion. Both LTC₄ and LTD₄ increased mucus secretion over baseline levels by a statistically significant level (p = < 0.05). LTD₄ was more potent than C₄ with relative potencies of 2500, 320, 630, and 500 based on hillock formation (a measure of secretion) at 1, 2, 3, and 4 minutes after injection. The overall difference in potency in this animal model of mucus production was LTD₄ > C₄ by 1000-fold.     

The effect of leukotrienes C4 and D4 on the microvasculature of guinea-pig skin

The effects of chemically-synthesised leukotrienes C₄ and D₄ (5(S) hydroxy-6(R)-δ-glutamylcysteinylglycinyl-7,9,11,14-eicosa-⁴tetraenoic acid, LTC₄; 5(S) hydroxy-6(R)-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, LTD₄) on the microvasculature have been measured in guinea-pig skin using [¹²⁵I]-albumin accumulation to measure plasma exudation and ¹³³Xe clearance to measure blood flow changes. As previously shown using biosynthetic material, LTD₄ caused vasoconstriction resulting in reduced blood flow. Similarly, LTC₄ was found to have vasoconstrictor activity but was more potent and had a steeper dose-response curve than LTD₄. There was no evidence of conversion of exogenous arachidonic acid to vaso-constrictor activity in the skin $\text{in vivo}$ (in the absence of another stimulus): intradermally injected arachidonic acid produced vasodilatation, but induced little change in blood flow in animals pretreated with indomethacin. The vasodilator effect of arachidonic acid is presumed to be due to conversion to either PGE₂ or PGI₂. These results suggest that cyclo-oxygenase is normally active in the skin, whilst lipoxygenase requires activation in some way. As reported in a previous study, LTD₄ induced plasma exudation when injected into the skin, but pronounced responses could only be induced by LTD₄ mixed with a vasodilator prostaglandin such as PGE₂. In contrast, LTC₄ induced no exudation when tested alone and little when PGE₂ was added. However, evidence was obtained that LTC₄ has some permeability-increasing activity which is marked by its potent vasoconstrictor activity.     

Formation of 1,24-24-trihydroxyvitamin D3 from 1,25-dihydroxyvitamin D3

Incubation of [26,27-³H₂]-25-hydroxyvitamin D₃ with kidney homogenates from rats fed a high (3%) calcium vitamin D-supplemented diet results in the production of a more polar metabolite which cochromatographs with 1,24,25-trihydroxyvitamin D₃. On the other hand, incubation with kidney homogenates from vitamin D-deficient or calcium-deficient rats did not produce the polar metabolite. Mitochondria but not microsomes carry out the reaction and evidence has been produced to demonstrate that the 1,24,25-trihydroxyvitamin D₃ can be produced in vivo from either 1,25-dihydroxyvitamin D₃ as previously reported.